Transgene-free Genome Editing in Plants

Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

Vinification without Saccharomyces: Interacting Osmotolerant and “Spoilage” Yeast Communities in Fermenting and Ageing Botrytised High-Sugar Wines (Tokaj Essence)

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as “spoilage yeasts“, ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L−1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Spatial organization in microbial range expansion emerges from trophic dependencies and successful lineages

Evidence suggests that bacterial community spatial organization affects their ecological function, yet details of the mechanisms that promote spatial patterns remain difficult to resolve experimentally. In contrast to bacterial communities in liquid cultures, surface-attached range expansion fosters genetic segregation of the growing population with preferential access to nutrients and reduced mechanical restrictions for cells at the expanding periphery. Here we elucidate how localized conditions in cross-feeding bacterial communities shape community spatial organization. We combine experiments with an individual based mathematical model to resolve how trophic dependencies affect localized growth rates and nucleate successful cell lineages. The model tracks individual cell lineages and attributes these with trophic dependencies that promote counterintuitive reproductive advantages and result in lasting influences on the community structure, and potentially, on its functioning. We examine persistence of lucky lineages in structured habitats where expansion is interrupted by physical obstacles to gain insights into patterns in porous domains.

Genetic segregation for male body coloration and female mate preference in the guppy

Objective The purpose of this study was to segregate the genetic lines responsible for the orange area of coloration in males and the response to orange coloration exhibited by females in the guppy ( Poecilia reticulata ) through artificial selection. This study is part of a project that uses QTL-seq to search for candidate genes involved in male orange coloration and female response to male coloration. We created two lines: high-selected lines of males having large areas of orange spots and of females with high response to male orange coloration; and low-selected lines of males having small areas of orange spots and of females with low response to male orange coloration.Results The male orange area and the female response became significantly different between high- and low-selected lines after three generations of artificial selection. This indicates that the differences in the frequencies of alleles at loci affecting the orange area and the female response between the lines increased over the generations through selection.

Land Invasion by the Mudskipper, Periophthalmodon septemradiatus, in Fresh and Saline Waters of the Mekong River

There has been a long-standing controversy about whether vertebrates emerged in the Paleozoic from marine or freshwater environments. Several hypotheses have proposed coastal, estuarine and riparian areas as sites of the transition. Here, we report the ecology of an amphibious fish Periophthalmodon septemradiatus, which we presume is in the process of niche expansion into terrestrial habitats from estuarine to freshwater environments along the Mekong River, Vietnam. Adult fish are highly terrestrial and have not been observed to venture into water during our survey. Courtship behaviour was observed, and fertilised eggs were recovered from burrows in both brackish and freshwater environments. The smallest fish collected at 12, 96, and 148 km from the river mouth were juveniles shortly after starting an amphibious life. These findings suggest reproduction in both brackish and freshwater environments. In contrast, otolith Sr:Ca ratio indicates larval hatching only in brackish water. Analysis of a 940-base pair (bp) segment of the mitochondrial cytochrome c oxidase subunit II and a 934-bp segment of the mitochondrial D-loop demonstrated no genetic segregation between populations. The fish may provide a unique opportunity to study how ambient salinity affects the biology and ecology of a living vertebrate during transition from water to land.

Genetic segregation for male body coloration and female mate preference in the guppy

Objective The purpose of this study was to segregate the genetic lines responsible for the orange area of coloration in males and the response to orange coloration exhibited by females in the guppy (Poecilia reticulata) through artificial selection. This study is part of a project that uses QTL-seq to search for candidate genes involved in male orange coloration and female response to male coloration. We created two lines: high-selected lines of males having large areas of orange spots and of females with high response to male orange coloration; and low-selected lines of males having small areas of orange spots and of females with low response to male orange coloration.Results The male orange area and the female response became significantly different between high- and low-selected lines after three generations of artificial selection. This indicates that the differences in the frequencies of alleles at loci affecting the orange area and the female response between the lines increased over the generations through selection.